RESUMEN
The ribosomal DNA (rDNA) represents a particularly unstable locus undergoing frequent breakage. DNA double-strand breaks (DSBs) within rDNA induce both rDNA transcriptional repression and nucleolar segregation, but the link between the two events remains unclear. Here we found that DSBs induced on rDNA trigger transcriptional repression in a cohesin- and HUSH (human silencing hub) complex-dependent manner throughout the cell cycle. In S/G2 cells, transcriptional repression is further followed by extended resection within the interior of the nucleolus, DSB mobilization at the nucleolar periphery within nucleolar caps, and repair by homologous recombination. We showed that nuclear envelope invaginations frequently connect the nucleolus and that rDNA DSB mobilization, but not transcriptional repression, involves the nuclear envelope-associated LINC complex and the actin pathway. Altogether, our data indicate that rDNA break localization at the nucleolar periphery is not a direct consequence of transcriptional repression but rather is an active process that shares features with the mobilization of persistent DSB in active genes and heterochromatin.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , ADN Ribosómico/genética , Regulación de la Expresión Génica/genética , ARN Largo no Codificante/metabolismo , Nucléolo Celular/metabolismo , Histonas/metabolismo , Recombinación Homóloga/genética , Membrana Nuclear/metabolismo , CohesinasRESUMEN
Gene duplications and transcriptional enhancer emergence/modifications are thought having greatly contributed to phenotypic innovations during animal evolution. Nevertheless, little is known about how enhancers evolve after gene duplication and how regulatory information is rewired between duplicated genes. The Drosophila melanogaster bric-a-brac (bab) complex, comprising the tandem paralogous genes bab1 and bab2, provides a paradigm to address these issues. We previously characterized an intergenic enhancer (named LAE) regulating bab2 expression in the developing legs. We show here that bab2 regulators binding directly the LAE also govern bab1 expression in tarsal cells. LAE excision by CRISPR/Cas9-mediated genome editing reveals that this enhancer appears involved but not strictly required for bab1 and bab2 co-expression in leg tissues. Instead, the LAE enhancer is critical for paralog-specific bab2 expression along the proximo-distal leg axis. Chromatin features and phenotypic rescue experiments indicate that LAE functions partly redundantly with leg-specific regulatory information overlapping the bab1 transcription unit. Phylogenomics analyses indicate that (i) the bab complex originates from duplication of an ancestral singleton gene early on within the Cyclorrhapha dipteran sublineage, and (ii) LAE sequences have been evolutionarily-fixed early on within the Brachycera suborder thus predating the gene duplication event. This work provides new insights on enhancers, particularly about their emergence, maintenance and functional diversification during evolution.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Elementos de Facilitación Genéticos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The evolutionarily conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Pol II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. But are these binary partnerships sufficient to mediate TF functions? We have previously established that the Med1 Mediator subunit serves as a cofactor of GATA TFs in Drosophila, as shown in mammals. Here, we observe mutant phenotype similarities between another subunit, Med19, and the Drosophila GATA TF Pannier (Pnr), suggesting functional interaction. We further show that Med19 physically interacts with the Drosophila GATA TFs, Pnr and Serpent (Srp), in vivo and in vitro through their conserved C-zinc finger domains. Moreover, Med19 loss of function experiments in vivo or in cellulo indicate that it is required for Pnr- and Srp-dependent gene expression, suggesting general GATA cofactor functions. Interestingly, Med19 but not Med1 is critical for the regulation of all tested GATA target genes, implying shared or differential use of MED subunits by GATAs depending on the target gene. Lastly, we show a direct interaction between Med19 and Med1 by GST pulldown experiments indicating privileged contacts between these two subunits of the MED middle module. Together, these findings identify Med19/Med1 as a composite GATA TF interface and suggest that binary MED subunit-TF partnerships are probably oversimplified models. We propose several mechanisms to account for the transcriptional regulation of GATA-targeted genes.
Asunto(s)
Proteínas de Drosophila/metabolismo , Factores de Transcripción GATA/metabolismo , Complejo Mediador/metabolismo , Animales , Sitios de Unión , Proteínas de Drosophila/genética , Drosophila melanogaster , Factores de Transcripción GATA/genética , Regulación de la Expresión Génica/genéticaRESUMEN
Genomic DNA is packed in chromatin fibers organized in higher-order structures within the interphase nucleus. One level of organization involves the formation of chromatin loops that may provide a favorable environment to processes such as DNA replication, transcription, and repair. However, little is known about the mechanistic basis of this structuration. Here we demonstrate that cohesin participates in the spatial organization of DNA replication factories in human cells. Cohesin is enriched at replication origins and interacts with prereplication complex proteins. Down-regulation of cohesin slows down S-phase progression by limiting the number of active origins and increasing the length of chromatin loops that correspond with replicon units. These results give a new dimension to the role of cohesin in the architectural organization of interphase chromatin, by showing its participation in DNA replication.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Cromatina/química , Proteínas Cromosómicas no Histona/fisiología , Empaquetamiento del ADN , Replicación del ADN , Proteínas de Ciclo Celular/análisis , Línea Celular , Proteínas Cromosómicas no Histona/análisis , Humanos , Interfase , Origen de Réplica , Fase S , CohesinasRESUMEN
Mitochondrial morphology varies according to cell type and cellular context from an interconnected filamentous network to isolated dots. This morphological plasticity depends on mitochondrial dynamics, a balance between antagonistic forces of fission and fusion. DRP1 and FIS1 control mitochondrial outer membrane fission and Mitofusins its fusion. This review focuses on OPA1, one of the few known actors of inner membrane dynamics, whose mutations provoke an optic neuropathy. Since its first identification in 2000 the characterization of the functions of OPA1 has made rapid progress thus providing numerous clues to unravel the pathogenetic mechanisms of ADOA-1.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , Apoptosis , ADN Mitocondrial/metabolismo , Metabolismo Energético , GTP Fosfohidrolasas/genética , Humanos , Fusión de Membrana , Mitocondrias/ultraestructura , Membranas Mitocondriales/metabolismo , Membranas Mitocondriales/ultraestructura , Mutación , Atrofia Óptica Autosómica Dominante/fisiopatologíaRESUMEN
The multi-subunit Mediator complex plays a critical role in gene expression by bridging enhancer-bound transcription factors and the RNA polymerase II machinery. Although experimental case studies suggest differential roles of Mediator subunits, a comprehensive view of the specific set of genes regulated by individual subunits in a developing tissue is still missing. Here we address this fundamental question by focusing on the Med19 subunit and using the Drosophila wing imaginal disc as a developmental model. By coupling auxin-inducible degradation of endogenous Med19 in vivo with RNA-seq, we got access to the early consequences of Med19 elimination on gene expression. Differential gene expression analysis reveals that Med19 is not globally required for mRNA transcription but specifically regulates positively or negatively less than a quarter of the expressed genes. By crossing our transcriptomic data with those of Drosophila gene expression profile database, we found that Med19-dependent genes are highly enriched with spatially-regulated genes while the expression of most constitutively expressed genes is not affected upon Med19 loss. Whereas globally downregulation does not exceed upregulation, we identified a functional class of genes encoding spatially-regulated transcription factors, and more generally developmental regulators, responding unidirectionally to Med19 loss with an expression collapse. Moreover, we show in vivo that the Notch-responsive wingless and the E(spl)-C genes require Med19 for their expression. Combined with experimental evidences suggesting that Med19 could function as a direct transcriptional effector of Notch signaling, our data support a model in which Med19 plays a critical role in the transcriptional activation of developmental genes in response to cell signaling pathways.
Asunto(s)
Drosophila , Discos Imaginales , Animales , Drosophila/genética , Activación Transcripcional , ARN Polimerasa II , Factores de Transcripción/genéticaRESUMEN
The GINS complex, originally discovered in Saccharomyces cerevisiae and Xenopus laevis, binds to DNA replication origins shortly before the onset of S phase and travels with the replication forks after initiation. In this study we present a detailed characterization of the human GINS (hGINS) homolog. Using new antibodies that allow the detection of endogenous hGINS in cells and tissues, we have examined its expression, abundance, subcellular localization and association with other DNA replication proteins. Expression of hGINS is restricted to actively proliferating cells. During the S phase, hGINS becomes part of a Cdc45-MCM-GINS (CMG) complex that is assembled on chromatin. Down-regulation of hGINS destabilizes CMG, causes a G1-S arrest and slows down ongoing DNA replication, effectively blocking cell proliferation. Our data support the notion that hGINS is an essential component of the human replisome.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Replicación del ADN , Anticuerpos , Línea Celular , Proliferación Celular , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/inmunología , Humanos , Subunidades de Proteína/inmunología , Subunidades de Proteína/metabolismo , Fase S/genéticaRESUMEN
BACKGROUND INFORMATION: Human OPA1 (optic atrophy type 1) is a dynamin-related protein of the mitochondrial IMS (intermembrane space) involved in membrane fusion and remodelling. Similarly to its yeast orthologue Mgm1p that exists in two isoforms generated by the serine protease Pcp1p/Rbd1p, OPA1 exists in various isoforms generated by alternative splicing and processing. In the present paper, we focus on protease processing of OPA1. RESULTS: We find that various mammalian cell types display a similar pattern of OPA1 isoforms [two L-OPA1 (long isoforms of OPA1) and three S-OPA1 (short isoforms of OPA1)] and that loss of the inner membrane potential, but not inhibition of oxidative phosphorylation or glycolysis, induces rapid and complete processing of L-OPA1 to S-OPA1. In isolated mitochondria, OPA1 processing was inhibited by heavy-metal chelators, pointing to processing by a mitochondrial metalloprotease. The pattern of OPA1 isoforms and its processing kinetics were normal in mitochondria devoid of the serine protease PARL (presenilins-associated rhomboid-like protein) - the human orthologue of Pcp1/Rbd1 - and in cells from patients carrying homozygous mutations in SPG7 (spastic paraplegia type 7), a gene encoding the matrix-oriented metalloprotease paraplegin. In contrast, OPA1 processing kinetics were delayed upon knock-down of YME1L (human yme1-like protein), an IMS-oriented metalloprotease. OPA1 processing was also stimulated during apoptosis, but inhibition of this processing did not affect apoptotic release of OPA1 and cytochrome c. Finally, we show that all OPA1 isoforms interact with Mfn1 (mitofusin 1) and Mfn2 and that these interactions are not affected by dissipation of DeltaPsim (inner mitochondrial membrane potential) or OPA1 processing. CONCLUSIONS: Metalloprotease-mediated processing of OPA1 is modulated by the inner membrane potential and is likely to be mediated by the YME1L protease.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Potencial de la Membrana Mitocondrial/fisiología , Metaloendopeptidasas/metabolismo , Metaloproteasas/metabolismo , Membranas Mitocondriales/enzimología , ATPasas Asociadas con Actividades Celulares Diversas , Células HeLa , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Interferencia de ARN/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The mitochondria are dynamic organelles that constantly fuse and divide. An equilibrium between fusion and fission controls the morphology of the mitochondria, which appear as dots or elongated tubules depending the prevailing force. Characterization of the components of the fission and fusion machineries has progressed considerably, and the emerging question now is what role mitochondrial dynamics play in mitochondrial and cellular functions. Its importance has been highlighted by the discovery that two human diseases are caused by mutations in the two mitochondrial pro-fusion genes, MFN2 and OPA1. This review will focus on data concerning the function of OPA1, mutations in which cause optic atrophy, with respect to the underlying pathophysiological processes.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Mitocondrias/metabolismo , Mitocondrias/patología , Atrofia Óptica Autosómica Dominante/patología , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Proteínas de Unión al GTP/metabolismo , Humanos , Proteínas Mitocondriales/metabolismo , Mutación/genética , Atrofia Óptica Autosómica Dominante/genética , Atrofia Óptica Autosómica Dominante/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Levaduras/metabolismoRESUMEN
The fission yeast Schizosaccharomyces pombe, widely used for studies of cell cycle control and differentiation, provides an alternative and complementary model to the budding yeast Saccharomyces cerevisiae for studies of nucleo-mitochondrial interactions. There are striking similarities between S. pombe and mammalian cells, in both their respiratory physiology and their mitochondrial genome structure. This technical review briefly lists the general and specific properties that are helpful to know when starting to use fission yeast as a model system for mitochondrial studies. In addition, advice is given for cell growth and genetic techniques, tips for disruption of genes involved in respiration are presented. and a basic differential centrifugation protocol is provided for the isolation of purified mitochondria that are suitable for diverse applications such as subfractionation and in vitro import.
Asunto(s)
Fraccionamiento Celular/métodos , Mitocondrias/metabolismo , Modelos Biológicos , Schizosaccharomyces/metabolismo , Citocromos/metabolismo , Genes Fúngicos , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrolloRESUMEN
The ability of DNA double-strand breaks (DSBs) to cluster in mammalian cells has been a subject of intense debate in recent years. Here we used a high-throughput chromosome conformation capture assay (capture Hi-C) to investigate clustering of DSBs induced at defined loci in the human genome. The results unambiguously demonstrated that DSBs cluster, but only when they are induced within transcriptionally active genes. Clustering of damaged genes occurs primarily during the G1 cell-cycle phase and coincides with delayed repair. Moreover, DSB clustering depends on the MRN complex as well as the Formin 2 (FMN2) nuclear actin organizer and the linker of nuclear and cytoplasmic skeleton (LINC) complex, thus suggesting that active mechanisms promote clustering. This work reveals that, when damaged, active genes, compared with the rest of the genome, exhibit a distinctive behavior, remaining largely unrepaired and clustered in G1, and being repaired via homologous recombination in postreplicative cells.
Asunto(s)
Mapeo Cromosómico , Roturas del ADN de Doble Cadena , Genoma Humano , Línea Celular , Análisis por Conglomerados , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Replicación del ADN/efectos de los fármacos , Replicación del ADN/genética , ADN Intergénico/genética , Fase G1/efectos de los fármacos , Fase G1/genética , Histonas/metabolismo , Humanos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Dominios Proteicos , ARN Interferente Pequeño/metabolismo , Recombinación Genética/efectos de los fármacos , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
Mitochondrial morphology is controlled by large GTPases, such as Msp1p, whose action on mitochondrial membranes is not yet understood. The sub-mitochondrial localization of Msp1p, the subject of ongoing controversies, was found to be within the intermembrane space. Overexpression of Msp1p led to aggregation of the mitochondrial network, while its downregulation resulted in fragmentation of this network. Mutations affecting the integrity of the Msp1p GTPase function had a dominant phenotype and induced mitochondrial fragmentation followed by mitochondrial DNA loss and cell death. These effects were not observed in cells deleted for Dnm1p, an actor in mitochondrial fission, suggesting that Msp1p is involved in the fusion of mitochondria.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Dinaminas/química , Dinaminas/metabolismo , Proteínas Fúngicas/metabolismo , Membranas Intracelulares/metabolismo , Mitocondrias/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Secuencias de Aminoácidos , ADN Mitocondrial/metabolismo , Dinaminas/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/metabolismo , Regulación Fúngica de la Expresión Génica , Estructura Terciaria de Proteína , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Mutations in the OPA1 gene are associated with autosomal dominant optic atrophy. OPA1 encodes a dynamin-related protein orthologous to Msp1 of Schizosaccharomyces pombe and Mgm1p of Saccharomyces cerevisiae, both involved in mitochondrial morphology and genome maintenance. We present immuno-fluorescence and biochemical evidences showing that OPA1 resides in the mitochondria where it is imported through its highly basic amino-terminal extension. Proteolysis experiments indicate that OPA1 is present in the inter-membrane space and electron microscopy further localizes it close to the cristae. The strong association of OPA1 with membranes suggests its anchoring to the inner membrane.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Membranas Intracelulares/enzimología , Mitocondrias/enzimología , Células 3T3 , Animales , Dinaminas , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Ratones , Microscopía Electrónica , Mitocondrias/metabolismo , RatasRESUMEN
Although both homologous recombination (HR) and nonhomologous end joining can repair DNA double-strand breaks (DSBs), the mechanisms by which one of these pathways is chosen over the other remain unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation-sequencing (ChIP-seq) to analyze repair of multiple DSBs induced throughout the human genome, we identify an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes and are targeted to HR repair via the transcription elongation-associated mark trimethylated histone H3 K36. Concordantly, depletion of SETD2, the main H3 K36 trimethyltransferase, severely impedes HR at such DSBs. Our study thereby demonstrates a primary role in DSB repair of the chromatin context in which a break occurs.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Recombinación Homóloga , Línea Celular , Cromatina/metabolismo , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , Recombinasa Rad51/metabolismo , Transcripción GenéticaRESUMEN
Eukaryotic chromosomes are condensed into several hierarchical levels of complexity: DNA is wrapped around core histones to form nucleosomes, nucleosomes form a higher-order structure called chromatin, and chromatin is subsequently compartmentalized in part by the combination of multiple specific or unspecific long-range contacts. The conformation of chromatin at these three levels greatly influences DNA metabolism and transcription. One class of chromatin regulatory proteins called insulator factors may organize chromatin both locally, by setting up barriers between heterochromatin and euchromatin, and globally by establishing platforms for long-range interactions. Here, we review recent data revealing a global role of insulator proteins in the regulation of transcription through the formation of clusters of long-range interactions that impact different levels of chromatin organization.
Asunto(s)
Cromatina/metabolismo , Regulación de la Expresión Génica , Animales , Factor de Unión a CCCTC , Proteínas de Unión al ADN/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Proteínas del Ojo/metabolismo , Humanos , Proteínas Represoras/metabolismoRESUMEN
Mitochondrial morphology depends on the equilibrium between antagonistic fission and fusion forces acting on mitochondrial membranes. Inactivation of fusion induces the loss of mtDNA. When both fusion and fission are simultaneously inactivated, the loss of mtDNA is alleviated, along with mitochondrial fragmentation. Mechanisms involved in mtDNA maintenance thus seem to depend on a coordinated regulation of fusion and fission forces. We have studied the role of the dynamin Msp1p, a fusion effector in mitochondrial morphology, in relation to the maintenance of mtDNA. Two hydrophobic regions of Msp1p, predicted to be transmembrane segments, were shown to anchor the long form of the protein into mitochondrial membranes, whereas the short form, lacking these two domains, behaved as a peripheral membrane protein. Both domains were essential for the fusogenic activity of Msp1p, but deletion of the second domain alone induced loss of mtDNA and thus lethality. Our results demonstrate that the role of Msp1p in the control of mitochondrial morphology is distinct from that required for genome maintenance, and that only the latter function is essential for cell viability. This parallels recent observations that have distinguished the role of OPA1, the human orthologue of Msp1p, in mitochondrial dynamics from that in cristae organization and apoptosis. Furthermore, our observations may contribute to our understanding of the pathological mechanisms resulting from mutations in OPA1 that give rise to the ADOA syndromes.
Asunto(s)
ADN Mitocondrial/genética , Dinaminas/metabolismo , Genoma Fúngico , Mitocondrias/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiología , Dinaminas/genética , Genes Letales , Membranas Mitocondriales/metabolismo , Isoformas de Proteínas , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Mitochondria are enveloped by two closely apposed boundary membranes with different properties and functions. It is known that they undergo fusion and fission, but it has remained unclear whether outer and inner membranes fuse simultaneously, coordinately or separately. We set up assays for the study of inner and outer membrane fusion in living human cells. Inner membrane fusion was more sensitive than outer membrane fusion to inhibition of glycolysis. Fusion of the inner membrane, but not of the outer membrane, was abolished by dissipation of the inner membrane potential with K+ (valinomycin) or H+ ionophores (cccp). In addition, outer and inner membrane fusion proceeded separately in the absence of any drug. The separate fusion of outer and inner membranes and the different requirements of these fusion reactions point to the existence of fusion machineries that can function separately.
Asunto(s)
Adenosina Trifosfato/metabolismo , Fusión de Membrana/efectos de los fármacos , Mitocondrias/ultraestructura , Membranas Mitocondriales/efectos de los fármacos , Antimicina A/farmacología , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Línea Celular , Desoxiglucosa/farmacología , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Proteínas Luminiscentes , Mitocondrias/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Oligomicinas/farmacología , Fosforilación/efectos de los fármacos , Valinomicina/farmacología , Proteína Fluorescente RojaRESUMEN
OPA1 encodes a large GTPase related to dynamins, anchored to the mitochondrial cristae inner membrane, facing the intermembrane space. OPA1 haplo-insufficiency is responsible for the most common form of autosomal dominant optic atrophy (ADOA, MIM165500), a neuropathy resulting from degeneration of the retinal ganglion cells and optic nerve atrophy. Here we show that down-regulation of OPA1 in HeLa cells using specific small interfering RNA (siRNA) leads to fragmentation of the mitochondrial network concomitantly to the dissipation of the mitochondrial membrane potential and to a drastic disorganization of the cristae. These events are followed by cytochrome c release and caspase-dependent apoptotic nuclear events. Similarly, in NIH-OVCAR-3 cells, the OPA1 siRNA induces mitochondrial fragmentation and apoptosis, the latter being inhibited by Bcl2 overexpression. These results suggest that OPA1 is a major organizer of the mitochondrial inner membrane from which the maintenance of the cristae integrity depends. As loss of OPA1 commits cells to apoptosis without any other stimulus, we propose that OPA1 is involved in the cytochrome c sequestration and might be a target for mitochondrial apoptotic effectors. Our results also suggest that abnormal apoptosis is a possible pathophysiological process leading to the retinal ganglion cells degeneration in ADOA patients.